Method of screening non-peptide, non-steroid invertebrate pheromone compounds having mitogen-activated protein kinase modulating activity and compositions containing the same

ABSTRACT

A method of screening pheromone compounds for mitogen-activated protein (MAP) kinase modulating activity and for employing such pheromone compounds in compositions suitable for the prevention or treatment of diseases, conditions, and symptoms thereof benefitting from modulation of MAP kinase activity.

FIELD OF THE INVENTION

The present invention is generally directed to compounds havingmitogen-activated protein kinase modulating activity, and to methods ofscreening such compounds and to compositions employing the same.Modulation of mitogen-activated kinase activity has been associated withthe treatment of a variety of medical diseases, conditions, and/orsymptoms thereof in warm-blooded animals including humans.

BACKGROUND OF THE INVENTION

Intercellular communication is essential for the development and normalfunction of all multicellular organisms. Cells communicate and respondto extracellular signals by utilizing various cellular mechanisms calledsignal transduction pathways. These pathways involve a series ofmolecular events culminating in the activation of effector mechanismsthat result in specific cellular responses. Certain disturbances insignal transduction pathways are involved in the pathogenesis of variousdiseases, conditions and symptoms thereof including cancer,cardiovascular disease, inflammation, autoimmune diseases (e.g.rheumatoid arthritis), neurodegenerative diseases (e.g. Alzheimer'sdisease), and other diseases including acquired immune deficiencysyndrome (AIDS) and the like.

One of the important signal transduction pathways is themitogen-activated protein (MAP) kinase pathway. This pathway isessential in cellular growth and differentiation. It is known thatblockage of the MAP kinase pathway suppresses tumor growth. In thissignal transduction cascade, sequential activation of kinases andsubsequent protein phosphorylation lead to the activation oftranscription factors at the DNA level.

The sequential activation of kinases (also known as the protein kinasecascades) is a common mechanism of signal transduction in many cellularprocesses. Mitogen-activated protein kinases are believed to include upto five levels of protein kinases that sequentially activate each otherthrough the process of phosphorylation. Among several mechanisms foractivating these cascades, one is believed to be initiated by a smallGTP binding protein which transmits the signal to the protein kinases.The signal is then transmitted down the cascade by enzymes at thevarious levels. The existence of multiple levels in each of the MAPkinase cascades is believed essential for signal amplification,specificity and tight regulation of the transmitted signals.

Nicholas S. Duesbery et al., Nature Medicine, Vol. 5/7 (July, 1999)report that tumor cells proliferate and spread throughout the body inapparent disregard of normal environmental cues. The inhibition of themitogen-activated protein kinase signal transduction pathway enablestumor cells to revert to a non-transformed phenotype and bypass thearrest of tumor growth in the body. It is also known that many oncogenesactivate the mitogen-activated protein kinase signal transductionpathway and it is this inappropriate activation that mediates thetransformed phenotype.

The pathophysiology of many diseases involves dysfunctionalintercellular signaling. As indicated above, in cancer, for example,some oncogenes encode signal transduction pathway proteins involved inthe regulation of self proliferation. Expression of these oncogenes mayinduce chronic activation that results in uncontrolled proliferation.Thus, tumor cells are effectively released from normal regulation andthereby proliferate, enabling the tumor to enlarge. In conditionsmanifesting unstable angina and myocardial infarction, plateletsaggregate and participate in the occlusion of coronary arteries byresponding to a variety of signals released by the rupture of anatherosclerotic plaque. In this particular case, platelets actuallyrespond appropriately to signals they are receiving, but they do so inan inappropriate place.

In autoimmune disease, signaling molecules produced by the processingand presentation of self-antigens activate cells of the immune system,and the activated immune cells respond by eliminating the offensivestimulus. Cells in the body that express the self-antigen are targetedfor destruction, and once the function of these normal cells iscompromised, symptoms of the autoimmune disease become evident.

Since the pathogenesis of many diseases can be traced to a dysfunctionin intercellular signaling, compounds which modulate the signal pathwaycan have utility in the treatment of a variety of such diseases.

Pheromones are a class of chemicals that are communicative betweenanimals of the same species and elicit stereotypical behavior andendocrine responses. Although there is a wide variety and a large numberof pheromone chemical structures, a single pheromone molecule has beenshown to have biological effects in non-associated species such as aspecies of insects and elephants. Some chemical similarities existbetween pheromones at different species.

The term “invertebrate pheromone” (or “pheromone”) in the context of thepresent invention should be understood as encompassing any chemicalcompound isolated from any invertebrate species which is produced anddischarged from glands and external ducts and functions by influencingother members of the same species in one of the ways known in the art,and which possesses mitogen-activating protein (MAP) kinase modulationactivity. One example of an invertebrate order are insects, one of thepredominant species of insects being Lepidoptera.

The terms “pheromone” or “pheromone compound” encompasses the naturalpheromones as well as synthetic compounds which display a similar MAPkinase modulating activity. Such synthetic compounds include variousderivatives of the natural pheromones, as well as analogs thereof.

The pheromone communication system involves the release of specificchemicals from a pheromone producer (emitter), the transmission of thesechemicals in the environment to a receiver, and the processing of thesignals to mediate the appropriate behavioral responses in the receiver.Some pheromone compounds are believed to activate the vomeronasal organ(VNO) which resides interior to the main olfactory epithelium (MOE) in ablind-ended pouch within the septum of the nose. In both locations, theVNO and MOE neuroepithelial dendrites terminate in specialized ciliacontaining specific receptors that bind odorants. This binding initiatesa cascade of enzymatic reactions that results in the production ofmessengers and the eventual depolarization of the cell membrane.

The signal transduction mechanisms through which pheromone compoundsexert their effect involve various pathways which are commonly found invarious other biological processes.

Applicants have investigated pheromone compounds as a class of chemicalsthat have potential modulating activity for the MAP kinase signaltransduction pathway. The potential of active pheromone compounds tomodulate (i.e. activate or inhibit) the MAP kinase signal transductionpathways provides compounds which may be used for the treatment of awide variety of diseases, conditions, and symptoms thereof as mentionedabove. For example, MAP kinase pathway inhibitors have been described aseffective in treating cancer and other proliferative diseases such aspsoriasis and restenosis as disclosed in A. J. Bridges et al., U.S. Pat.No. 5,525,625. MAP kinase pathway inhibitors have also been described asbeing effective in abolishing resistance to myocardial infarctioninduced by heat stress in M. Joyeux et al., Cardiovasc. Drugs Ther.,Vol. 14/3, pp. 337-343 (June, 2000). It is also known that MAP kinasepathway activators can desirably affect wound healing and tissue repair.

It would therefore be a significant advance in the art of treatingdiseases, conditions and symptoms thereof to identify pheromonecompounds possessing MAP kinase modulating activity. It would also be afurther advance in the art to identify pheromone compounds which haveMAP kinase modulating activity because of their potential for use intreating a variety of diseases, conditions, and symptoms thereofincluding cancer, autoimmune disease, psoriasis, restenosis as well aswound healing and tissue repair resulting from physical causesincluding, but not limited to, medical treatment such as chemotherapy.It would be a further advance in the art if pheromone compounds havingMAP kinase modulating activity could be effectively identified from alibrary of pheromone compounds.

SUMMARY OF THE INVENTION

The present invention is generally directed to the discovery thatcertain pheromone compounds possess mitogen-activated protein (MAP)kinase modulating activity. In one aspect of the present invention,there is provided a screening method for identifying compounds havingMAP kinase modulating activity from a library of pheromone compounds. Ina further aspect of the invention there is provided pharmaceuticalcompositions containing as an active agent at least one MAP kinasemodulator. Such composition have potential for use in the treatment of avariety of diseases, conditions, and symptoms thereof.

In a first aspect of the present invention, there is a provided a methodof screening compounds to obtain those having mitogen-activated protein(MAP) kinase modulating activity, comprising the steps of:

-   -   a) providing a biological model for screening of compounds        having MAP kinase modulating activity, the model being        predictive for MAP kinase modulating activity;    -   b) testing a group of invertebrate-derived non-peptide,        non-steroid pheromone compounds in the biological model to        determine the presence of MAP kinase modulating activity; and    -   c) selecting at least one of the pheromone compounds possessing        MAP kinase modulating activity in the model to obtain at least        one selected pheromone compound.

In another aspect of the present invention, there is a method ofselecting compounds having MAP kinase modulating activity, comprisingthe steps of:

-   -   a) providing data relating to the three-dimensional (3D)        structure of at least one pharmacophore of a compound known to        possess MAP kinase modulating activity;    -   b) providing a library of compounds, the library comprising at        least one pheromone compound selected from invertebrate-derived,        non-peptide, and non-steroid pheromones; and    -   c) analyzing the 3D structure of one or more compounds of the        library and selecting a compound having a domain with a 3D        structure at least substantially similar to the 3D structure of        the pharmacophore.

Optionally, the above selection method further comprises the step oftesting a selected compound in a relevant biological model which ispredictive of the desired MAP kinase modulating activity.

In a further aspect of the present invention, there is provided apharmaceutical composition comprising an effective amount of at leastone invertebrate derived, non-peptide, non-steroid pheromone compound orderivative thereof having mitogen-activated protein kinase modulatingactivity in combination with a pharmaceutically acceptable carrier.Methods of using the compositions to modulate mitogen-activated proteinkinase signal transduction pathways is also encompassed by the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention it has been discovered thatcertain pheromone compounds, derivatives and analogs thereof(hereinafter “pheromone compounds”) possess MAP kinase modulatingactivity and that such pheromone compounds are a source ofpharmaceutical agents which may be used for preventing and/or treatingdiseases, conditions or symptoms thereof. Compounds of the presentinvention having potential pharmaceutical activity may be selected firston their ability to modulate MAP kinase activity in the associatedsignal transduction pathway. Such target compounds may be screened froma library of pheromone compounds to provide compounds having the desiredpharmaceutical uses which involve MAP kinase activity.

“Modulating activity” is defined herein as activity which eitheractivates, inhibits or otherwise varies the action of MAP kinase and/orthe corresponding enzyme cascade along the MAP kinase signaltransduction pathway. The selection of compounds which either increasesor slows the action of MAP kinase and/or corresponding enzyme cascadealong the MAP kinase signal transduction pathway, may be carried outusing known assays in the art. Alternatively, it may also be possible,in accordance with the present invention, to separately screen compoundswhich increases or activates the MAP kinase activity in the associatedsignal transduction pathway, and to separately screen compounds whichcause a slowing or inhibiting effect on the MAP kinase activity using aseparate assay. In addition, compounds of the present invention may beselected directly which are useful in indications known to involve MAPkinase signal transduction pathway.

“Activators” as used herein include pheromone compounds which increaseor activate MAP kinase activity and/or a corresponding enzyme cascade ofthe MAP kinase signal transduction pathway.

“Inhibitors” as used herein include pheromone compounds which slow orinhibit MAP kinase activity and/or the corresponding enzyme cascade ofthe MAP kinase signal transduction pathway.

The term “therapeutic activity” is defined herein as any activity of acompound on specific target cells, tissue or organ, or activity whichachieves a specific effect within a body including prevention and/ortreatment of a disease, condition or symptom thereof, associated with orrelated to MAP kinase signal transduction pathway in warm-bloodedanimals including humans. The term “therapeutic activity” is furtherdefined as any activity which is manifested in relatively low levels orconcentration of the active compound, which is a concentration whichgives rise to a biological effect within the body, not through a generalsystemic effect, but rather through a specific effect on specifictargets within the body especially those responsive to modulation of theMAP kinase signal transduction pathway.

When the compounds are first selected on the basis of their ability tomodulate MAP kinase activity, the selection may be carried out using oneor more assays known in the art.

A biological model suitable for screening pheromone compounds for MAPkinase modulation activity may be based on a model which is known andacceptable in the literature for screening of such compounds to selectthose which have the desired activity. In some indications, theacceptable models are in vitro models, e.g. models involving testing ofthe effect of the compound on a cell or tissue culture. In otherindications, a relevant model having a predictive value is an in vivomodel involving laboratory animals, which may include rodents such asrats, mice or rabbits; xenograft models, e.g. an immune-compromisedmouse carrying human tissue; and higher order animals such as cats, dogsand even primates. Furthermore, the relevant model may at times be acombination of in vitro and in vivo models, or a combination of severalin vitro and/or several in vivo models. In addition, at times therelevant model may also be a model of non-living material such as amodel of isolated membranes, a variety of biochemical assays, and thelike.

In a preferred screening method, as set forth in Example 1 herein,pheromone compounds with MAP kinase modulating activity are identifiedusing Rat1 cells grown in cell culture dishes. Samples of the pheromonecompound are added to respective cell culture dishes containing the Rat1cells and incubated. A control group is established by treating aseparate group of cell culture dishes containing Rat1 cells with thesame solutions absent the pheromone compound. After incubation, the Rat1cells are stimulated with epidermal growth factor to stimulate MAPkinase activation in the Rat1 cells. Upon completion of the incubationperiod, the Rat1 cells in each of the culture dishes are separatelyprocessed to obtain the corresponding cell extract containing proteinsindicative of active kinase. The proteins are then separated using knownelectrophoresis techniques and prepared as a blot for the detection andmeasurement of active MAP kinase. The resulting blot is analyzed usingdensitometric techniques to measure the corresponding activity of thetested pheromone compound. The results obtained are compared to theresults obtained for the control to measure modulation of the active MAPkinase initially induced by EGF.

A pheromone compound having the desired MAP kinase modulating activitymay be selected on the basis of its three dimensional (3D) molecularstructure. The term “pharmacophore” is used herein as the structuraldomain of a compound which is associated with or related to a desiredpharmaceutical or biochemical activity. In this manner, the structure ofcompound having a known pharmaceutical activity may be used as astructural template for efficiently selecting compounds havingpotentially similar pharmaceutical activity. Once the compounds areselected based on their molecular structure, the selected compounds aretested in the relevant biological model as described above, to determinethe presence of the desired pharmaceutical activity.

In accordance with the method described above, the following compoundshave been observed to possess mitogen-activating protein kinasemodulation activity. MAP kinase activators include cis-7-tetradecenal,cis-7-tetradecenol, cis-7-dodecenyl ester, heneicosene-11-one,cis-6-heneicosene-11-one, cis-4-tridecenyl ester, cis-4-tridecen-1-ylester, 2-heptanone, cis-7-tetradecenyl ester, trans-5-decenyl ester,cis-2-methyl-7-octadecene, cis-9-heneicosene, trans-2,cis-13-octadecadienal, 14-methyl-cis-8-hexadecenal,2-methyl-3-butene-2-ol, trans-10-dodecenol, cis-9-tetradecenyl-formate,cis-9-tetradecenol, trans-3, cis-8, cis-11-tetradecatrienol,cis-7-tridecenol, cis-9-pentadecenol, and cis-9-undecenyl ester.Applicants have also discovered mitogen-activated protein kinaseinhibitors, namely, trans-3, cis-7-tetradecadienyl ester and trans-3,cis-8-tetradecadienyl ester. The ester of the above-identified compoundsmay be any ester, preferably an acetate.

The above-identified compounds were screened in accordance with themethods described above and thereby demonstrated the desired MAP kinaseinhibitory or activating activity.

The non-peptide, non-steroid invertebrate-derived pheromones of theinvention may be isolated from an invertebrate by any one of the methodsknown in the art. Alternatively, the pheromone compound of the presentinvention may also be chemically synthesized such as disclosed in U.S.Pat. No. 5,728,376 incorporated herein by reference. Many of theinvertebrate pheromone compounds including insect-derived pheromonecompounds may be synthesized through the fatty acid synthesis pathway.

The invertebrate-derived pheromone compounds of the present inventionhave a molecular weight typically less than about 500 Daltons, and moretypically less than about 300 Daltons.

Typically, the pheromone compound comprises a straight or branchedhydrocarbon chain of variable length (typically having a length of fromabout 6 to 30 carbon atoms, preferably from about 7 carbon atoms to 23carbon atoms, more preferably from about 9 carbon atoms to 21 carbonatoms). The hydrocarbon chain may comprise one or more double or triplebonds which may be located at any position in the chain, the doublebonds being in the cis or trans configuration. Typically, the sidechains in a branched main hydrocarbon chain may include alkyl groups,alkenyl groups, and/or alkynyl groups, each side chain comprising fromone to five carbon atoms. The main hydrocarbon chain may also compriseor be linked to a cycloalkyl or cycloalkenyl group having from about 3to 7 carbon atoms.

The main hydrocarbon chain may be substituted at any location of thechain by one or more functional groups including, for example, ahydroxyl, a ketone, an aldehyde, an epoxy group, a carboxylic acid, anester, a heterocyclic group, and an aromatic group.

In addition, the hydrocarbon chain of the pheromone compound may alsocomprise one or more additional groups which may, for example, beselected from ketones, halides (such as for example, F, Cl, Br, I),acetate esters, amines, thiols, thioesters, short chain alkyls (such asfor example, methyl, ethyl, propyl, butyl, pentyl). All of the abovemodifications may be carried out by known techniques utilized by one ofordinary skill in the art.

In accordance with the invention compounds which comprise modifiedfunctional groups in which, for example, an oxygen atom is replaced by asulfur atom, may also be used and fall within the meaning of the term“pheromone compound”as used herein. Thus, for example, a hydroxyl may bereplaced by a thiol, an ester or a thioester. Another example of amodification may be the replacement of a hydrogen atom by a halogenatom, such as, for example, a bromine atom. All of the abovemodifications may be carried out by known techniques utilized by one ofordinary skill in the art.

In accordance with the invention, derivatives or analogs of theinvertebrate pheromone compound may also be used if they substantiallymaintain the MAP kinase modulating activity of the parent pheromonecompound. The activity includes the desirable preventive or therapeuticactivity of the pheromone compounds observed to modulate MAP kinaseactivity along the MAP kinase signal transduction pathway.

A “derivative or analog” of a pheromone compound as used herein includesany compound having the basic structure of the invertebrate pheromone inwhich one or more functional groups are modified, but whichsubstantially maintains the desired activity of the unmodifiedpheromone. Such derivative or analog may include geometric isomers ofthe corresponding pheromone compound having the same or similar level ofMAP kinase modulating activity.

A derivative or analog which “substantially maintains” the activity ofthe unmodified pheromone, includes those displaying MAP kinasemodulating activity of a magnitude of at least 30%, preferably at least50% of the activity of the unmodified pheromone. Where the endbiological effect of the pheromone compound includes the preventionand/or treatment of a disease, condition or symptom, a derivative oranalog of the pheromone compound may be regarded as substantiallymaintaining the MAP kinase modulating activity, if it achieves a similarpreventive or therapeutic effect at a non-toxic concentration.

It will be understood that at times, the modification of a pheromonecompound to form a derivative or analog thereof, may achieve the desiredtherapeutic activity at the same time may result in the loss oralteration of one or more inherent properties associated with theunmodified pheromone compound.

The invention further provides a pharmaceutical composition suitable foradministration to a warm-blooded animal including humans, comprising asan active agent an effective amount of a pheromone compound as definedherein having a desired therapeutic activity associated with themodulation of MAP kinase activity, and a pharmaceutically acceptablecarrier.

The term “effective amount” as used herein includes an amount of theactive pheromone compound which provides a desirable preventive ortherapeutic effect in a warm-blooded animal including human afflictedwith a disease, condition or symptom related to MAP kinase activity inthe MAP kinase signal transduction pathway. It will be understood thatthe effective amount will depend on various factors such as, forexample, the nature of the indication for which the agent is used,characteristics of the treated warm-blooded animal, and mode ofadministration and the like, and will be routinely determined by one ofordinary skill in the art.

The effective amount of the pheromone compound will typically be in therange of from about 0.001 to 200 mg/kg/day, preferably from about 0.01to 20 mg/kg/day.

The pharmaceutical composition comprising at least one pheromonecompound having MAP kinase modulating activity may be formulated, forexample, by employing conventional solid or liquid vehicles or diluents,as well as pharmaceutical additives of a type appropriate to the mode ofdesired administration (for example, excipients, binders, preservatives,stabilizers, flavors, etc.) according to techniques such as those knownin the art of pharmaceutical formulation.

The present invention further provides a method of treating warm-bloodedanimals including humans afflicted with a disease, condition, orsymptoms which may benefit from modulation of MAP kinase activitycomprising administering to the warm-blooded animal an effective amountof a pheromone compound being selected from one or more suitablescreening methods disclosed herein and those that may be known in theart.

For use in the methods of the present invention associated with theprevention and/or treatment of diseases, conditions, and symptomsthereof, the pheromone compound may be administered to a warm-bloodedanimal including humans by one of a variety of administration modes,including, but not limited to oral, intravenous, intramuscular,transdermal, subcutaneous, topical, sublingual, rectal means, by nasalapplication, and the like. In addition, the pheromone compound may be avolatile substance and thus has the advantage of being administered byinhalation, resulting, in many cases in a very rapid response to theactive agent with relatively little, if any, side effects.

When the pheromone compound of the present invention is administeredorally, it may be administered in the form of a tablet, a pill, acapsule (e.g. a gelatin capsule), a powder or a pellet. Where a liquidcarrier is used, the oral preparation may be in the form of a syrup,emulsion, or soft gelatin capsule. Nasal administration may be by nasalinsufflation or as an aerosol, and internal administration such asrectal administration may be, for example, by use of a suppository. Fortopical administration the pheromone compounds may be, for example, inthe form of creams, ointments, lotions, solutions, gels or transdermalpatches.

The pheromone compounds of the invention may typically be administeredwith a pharmaceutically acceptable carrier which does not interfere withthe efficacy of the active pheromone compound. The carrier may beselected from a large number of carriers known in the art and the natureof the carrier will depend on the intended form of administration andindication for which the active agent is used.

Tablets, pills and capsules containing the pheromone compounds of theinvention may also include conventional excipients such as lactose,starch, and magnesium stearate. Suppositories may include excipientssuch as waxes and glycerol. Injectable solutions may comprise saline,buffering agents, dextrose, water glycerol, ethanol and solvents such aspropylene glycol, polyethylene glycol and ethanol. Such solutions mayalso comprise stabilizing agents and preservatives which are typicallyantimicrobial agents (such as chlorbutol, benzyl-alcohol, sodiumbenzoate, ascorbic acid, phenol, and the like) and antioxidants (such asbutylated hydroxy toluene, propyl gallate, sulfites, and the like).Enteric coatings, flavorings, and dyes and colorants may also be used.

At times, the pheromone compounds of the invention may be incorporatedwithin a liposome prepared by any of the methods known in the art. Inaddition, the pheromone compounds may be encapsulated in inertpolymerized particles such as, for example, nano particles,microspheres, microparticles, and the like known in the art.

The pheromone compounds of the invention may comprise a single activeagent or alternatively two or more such active agents which may exert anenhanced effect.

According to the methods of administration as encompassed by the presentinvention, the pheromone compounds may be administered separately or,alternatively, in combination with various other treatments administeredto the patient for the same or different disease, condition, or symptomthereof.

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the examples whichfollow, and from the claims, that various changes, modifications, andvariations can be made therein without departing from the spirit andscope of the invention.

The following examples are submitted for illustrative purposes only andare not intended to limit the invention as encompassed by the claimsforming part of the application.

EXAMPLE 1 Screening Method for the Identification of Pheromone CompoundsHaving MAP Kinase Modulating Activity

Rat 1 cells were grown in 6 cm cell culture dishes in a tissue cultureincubator at 37° C. in an atmosphere containing 5% carbon dioxide. Thecells were serum-starved for 16 hours. A solution containing from about5 to 500 ng/ml of a pheromone compound which may be a MAP kinaseactivator, inhibitor, or a compound which exhibits no MAP kinasemodulation activity, was added to the cell culture dishes and allowed tostand for about 15 minutes. A control group was established by adding tothe remaining cell culture dishes the same solution absent the pheromonecompound. After the 15 minute period, the Rat1 cells were stimulatedwith epidermal growth factor (EGF) (50 ng/ml) for about 15 minutes forone group and 30 minutes for a second group.

The Rat1 cells were then treated to remove the corresponding incubationmedium and then the cells were rinsed twice each with 5 ml of coldphosphate buffered saline (PBS) and once with 5 ml of cold homogenizingbuffer (Buffer H) containing 50 mM of β-glycerophosphate, (pH 7.3), 1.5mM of ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 1 mM ofethylenediaminetetraacetic acid (EDTA), 1 mM of dithiothreitol (DTT),0.1 mM sodium orthovanadate, 1 mM benzamidine, 10 μg/ml of aprotinin, 10μg/ml of leupeptin, and 2 μg/ml of pepstatin (2 μg/ml)+1% Triton-X-100.

The cells were harvested and lysed with 350 μl of cold Buffer H.Alternatively, the cells may be harvested and lysed withradioimmunoprotein assay buffer (RIPA buffer). Each extract was thencentrifuged at 15,000×g for 15 minutes at 4° C. The resultingsupernatants contained the protein extracts to be examined and weretransferred to fresh, pre-cooled test tubes and kept on ice.

The proteins were then separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred,by western blotting, onto a nitrocellulose membrane.

The blot produced above was blocked with 2% bovine serum albumin (BSA)in Tris-buffered saline/Tween-20 (TBST) for 1 hour. The blot was thenincubated with a monoclonal anti-active MAP kinase antibody obtainedfrom Sigma, and diluted according to the manufacturer recommendations.The blot was incubated overnight at 4° C. Alternatively, the blot mayalso be incubated for about 30 minutes at 37° C., or from one to twohours at room temperature.

The treated blot was placed in a flat container and washed at least 3times for 15 minutes each with TBST buffer at room temperature. The blotwas then incubated with alkaline phosphatase-conjugated goat anti-mouseIgG obtained from Jackson Laboratories, diluted according to themanufacturer recommendations in TBST buffer for 45 minutes at roomtemperature. The blot was then subjected to a second washing, repeated 3times, for 10 minutes each with TBST. An alkaline phosphatase (AP)detection technique was utilized to detect and measure the presence ofactive MAP kinase. Enhanced MAP kinase activity as compared to controlis evidence that the compound is a MAP kinase activator. Lowered MAPkinase activity as compared to control is evidence that the compound isa MAP kinase inhibitor. Unchanged MAP kinase activity as compared tocontrol is evidence that the compound is neither an activator nor aninhibitor of MAP kinase.

EXAMPLE 2 Demonstration of Pheromone Compounds Having MAP KinaseInhibiting Activity

Rat 1 cells grown in a series of cell culture dishes were prepared inthe same manner described above in Example 1. The cells wereserum-starved for 16 hours. Three groups of cell culture dishescontaining Rat 1 cells were each preincubated for about 30 minutes withsolution containing the pheromone compound, trans-3,cis-7-tetradecadienyl-acetate at a concentration of 10⁻⁴ M, with asolution containing the pheromone compound, trans-3,cis-8-tetradecadienyl-acetate at a concentration of 10⁻⁴ M, or a controlsolution absent any pheromone compound. After preincubation, all of theRat1 cells were stimulated with epidermal growth factor (EGF) (25 ng/ml)for about 15 minutes for one group and 30 minutes for a second group.The Rat1 cells were then washed with phosphate buffered saline (PBS) andlysed with RIPA buffer. The proteins were then separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probedwith anti-active-MAPK antibodies. The inhibitory effect of the pheromonecompound on MAPK activity was calculated as the percent EGF-inducedactivity as indicated by densitometry analysis as compared to thecontrol receiving no pheromone compound. The results are presented inTable 1 below. TABLE 1 Inhibition of MAP Kinase activity (% of Control)15 minute 30 minute Pheromone Compounds activation by EGF activation byEGF Trans-3,Cis-7-Tetradecadienyl- 75% 85% acetateTrans-3,Cis-8-Tetradecadienyl- 50% X acetateX - not available

As shown in Table 1 the pheromone compounds significantly inhibitedEGF-stimulated kinase activity as compared with the control.

EXAMPLE 3 Demonstration of Pheromone Compounds Having MAP KinaseActivating Activity Compared to Known Activating Agents of MAP Kinase

Rat 1 cells were grown in 6 cm cell culture dishes in a tissue cultureincubator at 37° C. in an atmosphere containing 5% carbon dioxide. TheRat1 cells were serum-starved for 16 hours. Three groups of the Rat1cells were then treated for 15 minutes with either epidermal growthfactor (EGF) (50 ng/ml), 12-O-tetradecanoylphorbol-13-acetate (TPA) (100nM), or with one of the pheromone compounds at one of two concentrationsidentified in Table 2 below. EGF and TPA are compounds which are knownto activate MAP kinase and therefore serve as a basis of comparing therelative activity of the pheromone compounds. The cells were then washedwith phosphate buffered saline (PBS) and lysed with radioimmunoproteinassay (RIPA) buffer. The proteins were then separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred ontoa nitrocellulose membrane and probed with anti-MAPK antibodies toproduce a blot. The MAP kinase activity for the EGF-treated cells, theTPA-treated cells and the pheromone compound-treated cells, was analyzedand calculated using densitometric analysis. The results shown in Table2 are presented as percent of EGF-stimulated active MAP kinase levels orTPA-stimulated active MAP kinase levels, respectively. For example, at aconcentration of 1×10⁻⁴ M, cis-7-tetradecenal, showed an MAP kinaseactivation that was 7% of the MAP kinase activation detected in ratcells stimulated by EGF. TABLE 2 Activity (% of Activity (% ofEGF-activation) TPA-activation) Conc. 1 Conc. 2 Conc. 1 Conc. 2Pheromone (1 × (1 × (1 × (1 × Compounds 10⁻⁴ M) 10⁻⁵ M) 10⁻⁴ M) 10⁻⁵ M)Cis-7-Tetradecenal 7 5 20 10 Cis-7-Tetradecenol 9 9 30 25Cis-7-Dodecenyl-acetate 7 8 20 17 Heneicosene-11-one 12  12  42 22Cis-4-Tridecen-1-yl-acetate 3 5  7 30 2-Heptanone 5 7 14 50Cis-7-Tetradecenyl-acetate 8 10  21 80 Trans-5-decenyl-acetate 1 4 X 18Cis-2-methyl-7-Octadecene 8 8 60 45 Cis-9-Heneicosene X 3 X 22Trans-2,Cis-13-Octadecadienal 4 8 30 40 14-methyl-Cis-8-Hexadecenal 10 3 75 15 2-methyl-3-butene-2-ol 5 X 35 X Trans-10-Dodecenol 2 10  10 60Cis-9-Tetradecenyl-formate 12  12  90 55 Cis-9-Tetradecenol 15  8 105 35 Trans-3,Cis-8,Cis-11- 6 5 10 50 Tetradecatrienol Cis-7-Tridecenol 12 9 80 60 Cis-9-Pentadecenol 14  6 95 30 Cis-9-Undecenyl-acetate 18  3115  10X-not available

As shown in Table 2, each of the pheromone compounds tested exhibitedincreased MAP kinase activity as compared with EGF or TPA.

EXAMPLE 4 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Parenteral Administration

4.5 grams (g) of beta-hydroxypropylcyclodextrin (beta-HPCD) were addedto 5.5 g of purified water and mixed together until the beta-HPCD wascompletely dissolved and a clear viscous solution was formed. 5 mg of apheromone compound (e.g. cis-7-tetradecenal, or cis-7-tetradecenol, ortrans-3, cis-7-tetradecadienyl acetate) of the present invention wasadded to the prepared HPCD solution and mixed under heating at 65-70° C.until the pheromone compound was completely dissolved. The resultingsolution was passed through a nylon membrane filter having a pore sizeof about 0.45 micron. The filtered solution was then allowed to cool.The thus prepared solution was diluted to a desired concentration using45% water solution of HPCD.

EXAMPLE 5 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Parenteral Administration

50 mg of hexadecanol was melted with 950 mg of Tween-80 in a water bath.The composition was then allowed to cool. Upon cooling, the gel-likecomposition was dissolved and an amount of a pheromone compound (e.g.cis-7-tetradecenal, or cis-7-tetradecenol, or trans-3,cis-7-tetradecadienyl acetate) of the present invention was dissolvedinto 99 ml of warm purified water. A transparent micellar solution wasthus formed suitable for parenteral administration.

EXAMPLE 6 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Parenteral Administration

A microemulsion composition was prepared by mixing 1800 mg of Tween-80,a non-ionic surfactant with 1500 mg of medium chain triglycerides oil(CRODAMOL™ TGCC). 100 mg of a pheromone compound (e.g.cis-7-tetradecenal, or cis-7-tetradecenol, or trans-3,cis-7-tetradecadienyl acetate) of the present invention was dissolvedinto the surfactant-oil mixture under slight heating at 45-50° C.Optionally, 200 mg of a cosolvent such as enthoxydiglycol (TRANSCUTOL™,Gattefosse, France) may be added for improved stability. The resultingsolution was filtered through a 0.22 micron membrane filter forsterility and the resulting solution was suitable for parenteralinjection.

EXAMPLE 7 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Parenteral Administration

A microemulsion composition was prepared by mixing 3600 mg of Tween-80,a non-ionic surfactant with 2700 mg of medium chain triglycerides oil(CRODAMOL™ TGCC). 100 mg of a pheromone compound (e.g.cis-7-tetradecenal, or cis-7-tetradecenol, or trans-3,cis-7-tetradecadienyl acetate) of the present invention was dissolvedinto the surfactant-oil mixture under slight heating at 45-50° C. Uponcooling, 300 mg glycol laurate (LABRASOL™) was added to the finalsolution and mixed thoroughly until dissolved. The solution was thenfiltered through a 0.22 micron membrane filter for sterility and theresulting solution was suitable for parenteral injection.

EXAMPLE 8 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Oral Administration

400 mg of a pheromone compound (e.g. cis-7-tetradecenal, orcis-7-tetradecenol, or trans-3, cis-7-tetradecadienyl acetate) of thepresent invention was admixed with 1.0 g of polyvinylpyrrolidone (PVPK-25™, BASF). The admixture was dissolved in 20 ml of ethyl alcohol USPgrade. The resulting solution was used for granulation of a mixture of4.6 g of dibasic calcium phosphate dihydrate (EMCOPRESS™, Mendell Co.)and 5.9 g of microcrystalline cellulose (AVICEL® pH102, FMC). Theresulting granulation was dried at 45° C. and passed through a stainlesssteel sieve (#16 mesh). The sieved granules were mixed with 0.1 g ofmagnesium stearate. 900 mg of the final granulation was encapsulated ina size 00 hard gelatin capsule, providing 30 mg of the pheromonecompound per capsule.

EXAMPLE 9 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Oral Administration

400 mg of a pheromone compound (e.g. cis-7-tetradecenal, orcis-7-tetradecenol, or trans-3, cis-7-tetradecadienyl acetate) of thepresent invention was admixed with 2.0 g of isopropyl palmitate, 4 g ofMIRJ®-52 (PEG-40 stearate, USP/NF grade), and 1.0 g ofpolyvinylpyrrolidone (PVP K-25™, BASF). The admixture was dissolved in30 ml of ethyl alcohol USP grade. The resulting solution was used forgranulation of a mixture of 5.6 g of calcium silicate (HIPERSORB™,Daminco) and 1.9 g of dibasic calcium phosphate dihydrate (EMCOPRESS™,Mendell Co.). The resulting granulation was dried at 45° C. and passedthrough a stainless steel sieve (#16 mesh). The sieved granules weremixed with 0.1 g of magnesium stearate. 750 mg of the final granulationwas encapsulated in a size 00 hard gelatin capsule, providing 20 mg ofthe pheromone compound per capsule.

EXAMPLE 10 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Suppository Use

125 mg of a pheromone compound (e.g. cis-7-tetradecenal, orcis-7-tetradecenol, or trans-3, cis-7-tetradecadienyl acetate) of thepresent invention was melted with 3.6 g of a suppository base comprisingcacao butter (99.6%) and butylated hydroxytoluene (0.4%). The meltedcomposition was mixed well with a spatula and then molded into four ovalsuppositoria using a suitable mold. Weighing 960 mg each, withsuppositoria containing about 30 mg of the pheromone compound.

EXAMPLE 11 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Topical Administration

An ointment comprising 5% a pheromone compound (e.g. cis-7-tetradecenal,or cis-7-tetradecenol, or trans-3, cis-7-tetradecadienyl acetate) of thepresent invention was prepared by mixing 500 mg of the pheromonecompound with 7.5 g of white petrolatum, 0.5 g lanolin and 1.5 g ofPOLAWAX™ (Emulsifying wax NF grade, Croda). The resulting mixture wasfurther mixed until cooling which resulted in an oleaginous ointment.

EXAMPLE 12 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Topical Administration

A water-in-oil emulsion cream containing 1% of a pheromone compound(e.g. cis-7-tetradecenal, or cis-7-tetradecenol, or trans-3,cis-7-tetradecadienyl acetate) of the present invention was prepared bydissolving 1 g of pheromone compounds in a mixture of 20 g of isopropylpalmitate and 10 g of POLAWAX™ (Emulsifying wax NF grade, Croda) at 70°C. to form a lipid phase. A water phase comprising 66 g of purifiedwater, 2.5 g of glycerin and 0.5 g of phenoxyethanol was heated to 70°C., then slowly added to the lipid phase, and vigorously mixed whilecooling. Upon cooling, a stable cream was obtained and packaged in anairtight container.

EXAMPLE 13 Formulation Containing Pheromone Compounds of the PresentInvention Suitable for Topical Administration

A clear gel preparation containing 0.5% of a pheromone compound (e.g.cis-7-tetradecenal, or cis-7-tetradecenol, or trans-3,cis-7-tetradecadienyl acetate) of the present invention was prepared byslowly adding 2.5 g of hydroxypropylcellulose (Klucel HF) to 37.0 g ofpurified water. The solution was heated to 70° C. The heated solutionwas mixed while allowing the temperature to fall to room temperatureresulting in a viscous gel as a water phase. Separately, 0.5 g of thepheromone compound of the present invention was dissolved in a mixtureof 40 g ethoxydiglycol (TRANSCUTOL™), 10 g of isopropyl myristate, and10 g of PLURONIC™ F-127 to form an organic phase. The water and organicphases were combined and slowly mixed until a uniform composition wasobtained. After degassing for 24 hours, a smooth clear gel was formed.

1. A method of screening compounds to obtain those havingmitogen-activated protein (MAP) kinase modulating activity, comprisingthe steps of: a) providing a biological model for screening of compoundshaving MAP kinase modulating activity, the model being predictive forMAP kinase modulating activity; b) testing a group ofinvertebrate-derived non-peptide, non-steroid pheromone compounds in thebiological model to determine the presence of MAP kinase modulatingactivity; and c) selecting at least one of the pheromone compoundspossessing MAP kinase modulating activity in the model to obtain atleast one selected pheromone compound.
 2. The method of claim 1 whereinthe MAP kinase modulating activity is MAP kinase inhibiting activity. 3.The method of claim 1 wherein the MAP kinase modulating activity is MAPkinase activating activity.
 4. The method of claim 1 further comprisingthe steps of: adding said pheromone compounds to a culture containingcells capable of expressing MAP kinase activity to form a test sample;adding a stimulator of MAP kinase to said test sample; measuring theamount of MAP kinase activity; and comparing the same to a controlsample containing no pheromone compound.
 5. The method of claim 1further comprising the steps of: treating a culture containing cellscapable of expressing MAP kinase activity with a stimulator of MAPkinase; adding said pheromone compounds to the culture; measuring theamount of MAP kinase activity; and comparing the same to a controlsample containing no pheromone compound.
 6. A pharmaceutical compositioncomprising an effective amount of a selected compound obtained by themethod of claim 1 in combination with a pharmaceutically acceptablecarrier.
 7. (canceled).
 8. A pharmaceutical composition comprising aneffective amount of a pheromone compound having MAP kinase modulatingactivity in combination with a pharmaceutically acceptable carrier. 9.The pharmaceutical composition of claim 8 wherein the effective amountis from about 0.1 to 200 mg/kg/day.
 10. The pharmaceutical compositionof claim 9 wherein the effective amount is from about 2.0 to 180mg/kg/day.
 11. The pharmaceutical composition of claim 8 wherein thepheromone compound has a molecular weight of less than about 500Daltons.
 12. The pharmaceutical composition of claim 8 wherein thepheromone compound has a straight or branched saturated or unsaturatedhydrocarbon chain having from about 6 to 30 carbon atoms.
 13. Thepharmaceutical composition of claim 12 wherein the pheromone compoundhas a straight or branched saturated or unsaturated hydrocarbon chainhaving from about 9 to 21 carbon atoms.
 14. The pharmaceuticalcomposition of claim 12 wherein the hydrocarbon chain is substitutedwith at least one member selected from the group consisting of an alkylgroup, an alkenyl group, an alkynyl group, a cycloalkyl group, acycloalkenyl group, a hydroxyl, a halide, an amine, a thiol, athioester, a ketone, an aldehyde, an epoxy group, a carboxylic acid, anester, an aromatic group, and a heterocyclic group.
 15. Thepharmaceutical composition of claim 8 wherein the pheromone compound isselected from the group consisting of cis-7-tetradecenal,cis-7-tetradecenol, cis-7-dodecenyl ester, heneicosene-11-one,cis-6-heneicosene-11-one, cis-4-tridecenyl ester, cis-4-tridecen-1-ylester, 2-heptanone, cis-7-tetradecenyl ester, trans-5-decenyl ester,cis-2-methyl-7-octadecene, cis-9-heneicosene, trans-2,cis-13-octadecadienal, 14-methyl-cis-8-hexadecenal,2-methyl-3-butene-2-ol, trans-10-dodecenol, cis-9-tetradecenyl-formate,cis-9-tetradecenol, trans-3, cis-8, cis-11-tetradecatrienol,cis-7-tridecenol, cis-9-pentadecenol, cis-9-undecenyl ester, trans-3,cis-7-tetradecadienyl ester and trans-3, cis-8-tetradecadienyl ester.16. The pharmaceutical composition of claim 15 wherein the ester is anacetate.
 17. The pharmaceutical composition of claim 8 wherein thepheromone compound inhibits MAP kinase activity.
 18. The pharmaceuticalcomposition of claim 17 wherein the MAP kinase inhibiting compound isselected from the group consisting of trans-3, cis-7-tetradecadienylester and trans-3, cis-8-tetradecadienyl ester.
 19. The pharmaceuticalcomposition of claim 8 wherein the pheromone compound activates MAPkinase activity.
 20. The pharmaceutical composition of claim 19 whereinthe MAP kinase activating compound is selected from the group consistingof cis-7-tetradecenal, cis-7-tetradecenol, cis-7-dodecenyl ester,heneicosene-11-one, cis-6-heneicosene-11-one, cis-4-tridecenyl ester,cis-4-tridecen-1-yl ester, 2-heptanone, cis-7-tetradecenyl ester,trans-5-decenyl ester, cis-2-methyl-7-octadecene, cis-9-heneicosene,trans-2, cis-13-octadecadienal, 14-methyl-cis-8-hexadecenal,2-methyl-3-butene-2-ol, trans-10-dodecenol, cis-9-tetradecenyl-formate,cis-9-tetradecenol, trans-3, cis-8, cis-11-tetradecatrienol,cis-7-tridecenol, cis-9-pentadecenol, and cis-9-undecenyl ester.
 21. Thepharmaceutical composition of claim 20 wherein the ester is an acetate.22-27. (canceled).
 29. A method of selecting compounds having MAP kinasemodulating activity, comprising the steps of: a) providing data relatingto the three-dimensional (3D) structure of at least one pharmacophore ofa compound known to possess MAP kinase modulating activity; b) providinga library of compounds, the library comprising at least one pheromonecompound selected from invertebrate-derived, non-peptide, andnon-steroid pheromones; and c) analyzing the 3D structure of one or morecompounds of the library and selecting a compound having a domain with a3D structure at least substantially similar to the 3D structure of thepharmacophore.
 30. The method of claim 29 wherein the MAP kinaseactivity is MAP kinase inhibiting activity.
 31. The method of claim 29wherein the MAP kinase activity is MAP kinase activating activity. 32.The method of claim 29 further comprising the steps of: providing abiological model for screening of compounds having MAP kinase modulatingactivity, the model being predictive for MAP kinase modulating activity;testing the selected compounds in the biological model for determiningpresence of MAP kinase modulating activity in tested pheromone compoundthereof; and selecting at least one of said compounds showing MAP kinasemodulating activity to obtain at least one selected compound.
 33. Apharmaceutical composition comprising an effective amount of a selectedcompound obtained by the method of claim 29 in combination with apharmaceutically acceptable carrier.
 34. A method of modulating MAPkinase activity comprising administering to a warm-blooded animal aneffective amount of the composition of claim
 33. 35. The method of claim22 wherein the disease, condition, or symptom thereof is selected fromthe group consisting of cancer, autoimmune disease, psoriasis,restenosis, and cardiovascular disease.